Gain-of-function study reveals the pleiotropic roles of serine protease HtrA inBorrelia burgdorferi

Abstract

ABSTRACTHigh-temperature requirement protease A (HtrA) is a family of serine proteases degrading misfolded and damaged proteins that are toxic to bacteria. The Lyme disease agentBorrelia burgdorferiencodes a single HtrA (BbHtrA). Previous studies have shown that BbHtrA is a key virulence determinant ofB. burgdorferias a deletion mutant ofhtrA(ΔhtrA) fails to establish infection in mice. However, previous complementation could only restore protein expression but not infectivity in mice. In this report, we first identify the native promoter of BbHtrA which allows us to construct a fully complementedΔhtrAstrain. Follow up promoter activity analysis reveals that BbHtrA is likely dually regulated by the house keeping sigma factor RpoD and the alternative sigma factor RpoS. TheΔhtrAmutant exhibits growth defect upon entering the mid-log to stationary phase especially at high temperatures. Microscopic analysis further demonstrates that the absence ofhtrAinduces extensive cell death. Additionally, theΔhtrAmutant has defects in cell locomotion as the expression of several key chemotaxis proteins are significantly downregulated. Cryo-electron tomography imaging ofhtrAmutant further reveals that deletion ofhtrAdisrupts flagellar homeostasis. The failure ofΔhtrAto establish an infection in mice is likely due to repressed expression of BosR and RpoS at the transcriptional level which ultimately causes dysregulation of the RpoS-induced virulence factors. Collectively, we conclude that the expression ofhtrAis finely tuned which is critical for its pleiotropic roles in the regulation of motility, stress response, and virulence gene expression inB. burgdorferi.IMPORTANCELyme borreliosis is the most commonly reported vector-borne illnesses in the United States, which is caused byBorrelia burgdorferi.As the enzootic pathogen alternates between the tick vector and mammalian hosts, adaptation to drastically different growth milieu is imperative to its survival. Hence, robust alteration of gene expression and proper quality control on protein synthesis and turnover are pivotal for its fitness. The family of HtrA serine proteases is mainly responsible for the maintenance of protein homeostasis particularly under stressful conditions. The significance of this report is to decode how BbHtrA contributes to the fitness ofB. burgdorferi. BbHtrA is essential for mammalian host infection but little is known about its regulatory mechanism as well as its contribution to the virulence ofB. burgdorferi. By deciphering the regulatory elements involved in the expression of BbHtrA, we are one step closer to comprehending its significance in the pathophysiology ofB. burgdorferi.